Cytotoxic Effects of Different Extracts and Latex of Ficus carica L. on HeLa cell Line.

It has been reported that latex and extracts of different species of Ficus are cytotoxic to some human cancerous cell lines. In this study, cytotoxicity of fruit and leaf extracts as well as the latex of Ficuscarica L. on HeLa cell line were evaluated. ethanolic extracts of leaves and fruits were prepared through percolation and ethyl acetate and dichloromethane extracts were prepared by reflux method. Cytotoxic effects of these extracts and latex against HeLa cell line were then examined. Briefly, He Lacells were seeded at 2 × 10(4) cells/mL in 96-well plates. After 24 h incubation at 37(°)C, the cells were treated with different concentrations of the extracts or latex. The viability of the cells was determined by the reduction of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) from formazan following 48 h incubation and the absorbance was measured at 540 nm using an ELISA plate reader. The results indicated that the latex and different extracts of Ficus carica could reduce the viability of the He Lacells at concentrations as low as 2 µg/mL in a dose dependent manner. The approximate IC50 values of the ethanolic, ethyl acetate and dichloromethane extracts of the leaves and fruits were 10, 19, 12 µg/mL and 12, 12, 11.5 µg/mL, respectively. The IC50 for the latex was about 17 µg/mL.


Introduction
In recent years, there has been a global trend towards the use of natural phytochemicals present in natural products such as fruits, vegetables, and their extracts. The leaf extracts of Ficus racemosa (4) and Ficus hispida hepatoprotective activity against carbon tetrachloride and paracetamol induced hepatotoxicity in rats. The methanol extract of the leaves of Ficus carica was recently shown to possess hepatoprotective activity in rats with liver damage induced by carbon tetrachloride at an oral dose of 500 mg/Kg by lowering the serum levels of aspartate aminotransferase, alanine aminotransferase, total serum bilirubin, and malondialdehyde equivalent, an index of lipid peroxidation of the liver (3).
Fig tree latex was used traditionally to treat wart (6). The Fig tree latex therapy of warts was recently compared with cryotherapy which duration therapy, no reports of any side-effects, ease-of-use, patient compliance, and a low recurrence rate (7) .The latex offers some other therapeutic effects such as anti-Herpes Simplex Virus (HSV)-1 (8), anthelmintic (9), anti mutagenic (10), antioxidative (11) and cytotoxic (12) activities.
More recent investigations indicated that different parts of Ficus species like fruit, stem, leaf and latex contain active ingredients like triterpenoides which have antioxidant and cytotoxic activities (19,20,21). For instance, triterpenes which were isolated from the aerial roots of Ficus microcarpa were cytotoxic to HONE-1, KB, and HT29 cell lines with IC 50 cytotoxic activity of fruit and leaf extracts and the latex of Ficus carica were evaluated.

Materials
The plants were collected from the Ficus Garden of Isfahan University of Medical Ficus carica. A sample (herb. no 1778) was kept in the herbarium of the department of pharmacognosy. The fruits and leaves were air dried and the latex was kept at Tripan blue, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased black cervix carcinoma, epithelioid) cells were purchased from Pasteur Institute of Iran (in Tehran). Roswell Park Memorial Institute (RPMI)-1640 culture medium, Fetal Bovine Serum (FBS), or Fetal Calf Serum (FCS), sodium pyruvate, penicillin/ streptomycin and trypsin-EDTA were purchased from Gibco (Gibco, Scotland). The absorbance was measured at 540 USA).

Extraction methods
Ethanolic extracts of the fruits and the leaves were prepared by percolation with 70 aqueous ethanol while ethyl acetate and dichloromethane The solvents evaporated using a rotary evaporator. The dry extracts were then used for cytotoxic assays. The latex was used directly Sample and culture medium preparations consisted of 5.2 g RPMI powder, 1 g of sodium inactivated fetal calf serum (FCS) in deionized water (24). The completed media was sterilized to 7.4 using concentrated HCl or NaOH and kept 2 -95% air prepared by dissolving 20 mg of the dry extracts total volume. The stock solutions were then appropriately diluted with the medium and suspensions in order to reach 2, 5, 10, 20, 50, and

In-vitro cytotoxicity assay
The cytotoxic effects of different extracts and the latex of Ficus carica were determined by a rapid colorimetric assay using MTT. The results were compared with untreated control (25). In this assay, mitochondrial succinic dehydrogenase enzyme of viable cells would metabolically alter the yellow soluble MTT salt into a blue insoluble formazan product. The blue solid could be dissolved in DMSO and measured spectrophotometrically (26). the cells (5 x 10 4 seeded in 96-well microplates and incubated for 2 ). Then, 20 were added and the microplates were further incubated for 48 h at the same condition. Taxol negative control while the blank wells consisted the cell survival, each well was then incubated phosphate buffer solution) for 3 h. Afterwards, the media in each well was gently replaced with dissolve the formazan crystals. The absorbance of each well was measured at 540 nm using an was constructed and used for the calculation of percent cell survival. The percent cell survival was taken as 100% for the negative control. The percentage of cell viability was calculated using the following formula:

Statistical analysis
The results are the mean of three triplicate experiments. SIGMASTAT (Jandel Software, San Raphael, CA) was used for statistical analysis. Analysis of variance (ANOVA) was used to see the differences amongst various

Results and Discussion
The results of cytotoxic assays of different    Table 1. activities of Ficus latex was performed by Ulman et al. (27). The cytotoxic effects may reside mostly in the resin of the latex of Ficus species, while the other ingredients of different parts of the plant may also contribute to these activities.
In the present work, cytotoxic effects of ethanol, ethyl acetate and dichloromethane extracts of Ficus carica and the crude latex were evaluated. According to the presence of different active ingredients in Ficus spp. with diverse solubilities, it seems that the ethanolic extracts may contain more polar ingredients like carbohydrates, proteins, resins and glucosylsitosterols (21,22,23) while the less polar solvents, ethyl acetate and dichloromethane, could extract less polar materials like triterpenoids, present in the leaves and fruits, some of which have cytotoxic potentials (12). The results indicated that all extracts from the fruits, leaves and the latex have moderate considerable difference, with approximate IC 50 could be due to the partial solubility of different active ingredients in the employed solvents.
These results are almost in accordance with the results obtained by Chiang et al. who separated some cytotoxic triterpenes from the aerial roots of Ficus microcarpa using moderate polarity solvent systems. The separated triterpenes were cytotoxic to HONE-1, KB and HT29 having IC 50 (22). On the other hand, 6-O-acyl---D-glucosyl--sitosterols which were provided by successive extractions with different polar solvents were also cytotoxic to a panel of cell lines including 50 values (12). The similar cytotoxic effects of the extracts and the latex could be due to the use of crude latex in our studies.